The overall objective of this proposal is to develop an innovative toxicogenomics approach for defining critical elements in cellular gene expression cascades that confer differential effects across species following exposure to physical or chemical agents. A new approach is required since traditional toxicogenomic methods have not reliably addressed cross-species differences in toxicity and have primary relied on correlative evidence to associate transcriptional changes with a pathological end point. The missing component is a mechanistic understanding of the cause-and-effect relationships contained within the lists of altered genes and the resulting toxicological outcome. The overarching hypothesis for the proposed research is that defining the cascading network of gene expression changes is essential for understanding the toxicity of chemicals and predicting their cross-species conservation of response. These changes include the interrelationships between the primary, secondary, and tertiary gene expression responses. This hypothesis will be tested using unique combination of genomic tools that can dissect these transcriptional responses following exposure to a prototypical hepatotoxicant, phenobarbital that has different carcinogenic activity in rodents and humans. The specific aims of this proposal are: (1) test the hypothesis that phenobarbital will cause temporally resolvable changes in gene expression in primary mouse and human hepatocytes through time-course gene expression studies using whole-genome microarrays; (2) test the hypothesis that the gene expression cascade for phenobarbital can be delineated by individually blocking key regulatory genes induced at earlier time points using RNA interference and measuring the effect on down-stream changes in gene expression; (3) test the hypothesis that specific secondary gene expression responses related to early cell proliferation events exist in mouse hepatocytes, but not in human hepatocytes. By determining the interrelationships between the secondary transcriptional responses and the primary regulatory genes, we will be able to mechanistically link the proliferative subnetwork to the primary event or receptor responsible for cross-species differences. Through this study we believe a novel approach to toxicology will be developed that allows comparisons of the signaling cascades across species to predict the conservation of the toxic response as well as provide new insights into the mechanism of action for many chemical agents.